ABSTRACT Recent advances in pluripotent stem cell technology have enabled generation of neuronal cell lines and cerebral organoids from human embryonic stem cells (hESCs) as well as human induced pluripotent stem cells (hiPSCs) derived from peripheral tissues. These organoids are self-assembled, 3D cellular structures that resemble early developmental stages of the human brain opening unprecedented opportunities for investigation of human neuronal network-level dysfunction underlying developmental brain disease. However, the lack of the natural brain microenvironment in cultured organoids can influence the phenotype and maturation of the reprogrammed neurons. To mitigate this limitation, we recently transplanted human cerebral organoids into the mouse brain and demonstrated their differentiation and vascularization using 2- photon imaging through cranial ?windows? made of glass. In the proposed study, we will replace these windows with optically transparent graphene electrode microgrids, developed by members of our team, to enable multimodal longitudinal monitoring and interrogation of neuronal activity in the graft and the surrounding host neuronal circuits. The in vivo organoid transplantation and graphene electrode arrays are existing technologies providing Scientific Premise to the proposed project ? without these parts in place, we would be building a bridge too far. Our current goal is to combine these technologies creating a synergistic and transformative result. Critically, this combination will allow examination of cell-type specific spiking (detected with 2-photon imaging) referenced to large-scale network events arising either from the organoid or the host cortex (detected as Local Field Potentials, LFPs, by a graphene electrode grid). We know from our prior work that organoids achieve sufficient laminar organization and synaptic connectivity to generate LFPs, adding to the Scientific Premise. We will engineer implantable graphene devices that would adhere to the cortical surface and the organoid to enable stable, longitudinal recordings, imaging, and photostimulation in mice transplanted with human cortical organoids (Aim 1). Then, we will provide a proof-of-principle demonstration of the unique advantage of this multimodal technology for studying the evolution of organoid activity during its maturation in vivo. To this end, we will focus on participation of specific cell types in LFP events originating from either the organoid or the host (Aim 2). All experiments will be performed in awake mice without confounds of anesthesia. Since transplantation of human cerebral organoids in the mouse brain is still in infancy, this project will deliver a much needed tool for comprehensive functional assessment of this novel biological model system. Further along the road (outside the current scope), this model system will find its use for investigating aspects of human brain development and developmental disorders.